RESUMEN
Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 - REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.
Asunto(s)
Trastornos de la Coagulación Sanguínea/patología , Plaquetas/patología , Regulación de la Expresión Génica , Células Madre Pluripotentes Inducidas/patología , Megacariocitos/patología , Mutación , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Trastornos de la Coagulación Sanguínea/genética , Trastornos de la Coagulación Sanguínea/metabolismo , Plaquetas/metabolismo , Diferenciación Celular , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Megacariocitos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fenotipo , Mapas de Interacción de Proteínas , Proteoma/análisis , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismoAsunto(s)
Susceptibilidad a Enfermedades , Trastornos Hemorrágicos/etiología , Mutación Missense , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Alelos , Expresión Génica , Estudios de Asociación Genética , Genotipo , Trastornos Hemorrágicos/sangre , Trastornos Hemorrágicos/diagnóstico , Humanos , Linaje , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.